- Email: [email protected]
Application of biosensors for diagnostic analysis and bioprocess monitoring
Sensors and Actuators B 65 Ž2000. 26–31 www.elsevier.nlrlocatersensorb
Application of biosensors for diagnostic analysis and bioprocess monitoring Jianguo Liu ) , Gaoxiang Li Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People’s Republic of China State Key Laboratory of Transducer Technology, People’s Republic of China Received 30 July 1998; accepted 8 January 1999
Abstract A biosensor based on chemiluminescence for determination of serum uric acid and two biosensors for glutamate- and penicillin-fermentation process monitoring and controlling have been developed. The preparations and some properties of these sensors have been described in detail. The sensor methods described have been compared with routine methods used and the good coincidences have been obtained. q 2000 Elsevier Science S.A. All rights reserved. Keywords: Uric acid sensor; Chemiluminescence; Glutamate fermentation process control; ENFET sensor
1. Introduction The serum uric acid concentration is an important index for clinical diagnosis of gout, leukemia toxemia of pregnancy and severe renal impairment. In the enzyme electrode research w1x, we found out that the sensitivity of the O 2 electrode did not meet the demand of serum uric acid measurement, especially when the concentration was below 3 mgrdl. The high sensitivity of luminol chemiluminescence can overcome this drawback. Biosensors for fermentation process control hold much promise for monitoring many of the important compounds involved in fermentation. These include glucose, glutamate, ethanol, organic acid, and various gases w2–4x. Recently, two biosensors for fermentation process control have been developed in our laboratory. One is the model on-line monitoring system and the other is the batch-type ENFET sensor. This paper introduces three biosensors developed in our laboratory recently.
)
Corresponding author. Fax: q86-10-62560912.
2. A chemiluminescence sensor for serum uric acid assay 2.1. Experiments and results 2.1.1. Principle of the assay The method was based on following reaction sequence: uric acid q O 2
™
uricase
allantoinq H 2 O 2 q CO 2
™
Ž 1.
ferricyanide
H 2 O 2 q luminol
aminophthalate anion q photonq N2 q 2H 2 O
Ž 2.
Reaction Ž1. is highly specific for uric acid. However, reaction Ž2. is catalyzed by enzyme Žperoxidase. or metal ions possessing an oxidation state w5x. Provided all the conditions are constant, the emitted photon intensity is proportional to uric-acid concentration in the serum. 2.1.2. Preparation of the chemiluminescent sensor Uricase ŽEC 1.7.3.3. was immobilized on the alkylamined pore glass with glutaraldehyde w6x. The immobilized enzyme was packed in a small plastic tube to build the enzyme reaction column. Fig. 1 indicates the flowthrough diagram of the sensor. A certain volume of serum sample met the carrier buffer through the valve injector,
0925-4005r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved. PII: S 0 9 2 5 - 4 0 0 5 Ž 9 9 . 0 0 4 3 1 - 1
J. Liu, G. Li r Sensors and Actuators B 65 (2000) 26–31
27
Fig. 1. Diagram of FIA for determination of uric acid using uricase-chemiluminescence sensor system.
then flowed into the immobilized enzyme reactor column. After enzymatic reaction, the mixture joined the luminol and ferricyanide solution in the reaction cell. The photon generated by chemiluminescent reaction was detected by a photodiode attached to the face of the reaction cell. The photon intensity was shown on the window of the photoelectric generator. 2.1.3. Response time The response time of the sensor to various concentrations of serum uric acid with a 17-ml sample volume was short. The photon intensity of the reaction reached its peak value about 47 s after injection. It required about 1.5 min for every sample measurement. 2.1.4. Linearity of the working curÕe Typical linearity of the working curve was from 1 up to 20 mgrdl Ž r s 0.9999; Fig. 2.. This linear range was wider than that obtained by the electrochemical electrode method w1x and the result reported by Tabata et al. w7x. 2.1.5. Precision and reproducibility Pooled serum samples with low and high contents of uric acid were analyzed 20 times Žwithin a day. or 20 times for 10 days Žday to day.. The imprecision within a
Fig. 2. Calibration curve for uric acid determination using uricase-chemiluminescence sensor system.
day was below 5%, and the day-to-day imprecision was below 8%, which were satisfactorily precise and reproducible for clinical analysis. 2.1.6. Operational stability The operational stability of the sensor depends on the stability of the immobilized uricase column. Used at room temperature Ž25–328C. and stored at 48C, the immobilized uricase column retained 94% of its original activity even after 2000 runs for 5.5 months of continual usage. No decrement of the photon intensity was observed. 2.1.7. Comparison with other method Sixty-one serum samples of high and low uric acid content were measured by the present method, and the standard colorimetric method routinely used in clinical labs employing the enzymatic kit ŽShanghai, China. performed on a Hitachi 7150 autoanalyzer. As shown in Fig. 3, excellent agreement was obtained. The calculated regression line and correlation coefficient were Y s 0.4 q 0.938 X Ž X data measured by enzymatic kit; Y data measured by present method. and r s 0.9909, respectively.
Fig. 3. Correlation and regression line for serum uric acid measurement assayed by uricase-chemiluminescence-sensor method against enzymatic kit method. X, data assayed by enzymatic kit method. Y, data assayed by the sensor method.
J. Liu, G. Li r Sensors and Actuators B 65 (2000) 26–31
28
3. An on-line monitoring system The purpose of this study was to construct a model on-line system for fermentation process control. The on-line monitoring of glutamate fermentation process was chosen as the target of the study. During the fermentation process, the concentration of glucose Žsubstrate. and glutamate Žproduct. are very important parameters, which reflect the development of the fermentation process. 3.1. Experiments and results 3.1.1. Principle of the measurement The measurement is based on the following reaction sequences:
™
glucose oxidase
Glucoseq O 2 q H 2 O
Glucono-D-lactone q H 2 O2
™
Ž 3.
glutamate oxidase
2-L-glutamateq O 2 q H 2 O
2-oxoglutarate q 2NH 3 q H 2 O 2
nylon tube reactors, hydrogen peroxide electrode, analog– digital converter, recorder and feedback control unit. Driven by the electronic micromotor pump, the fermentation broth passes through the sampling unit, which prevents the microorganisms and insoluble materials from passing into the downstrain analytic system, and flows through the dilution unit and the injection valve, ensuring the same sample volume Ž15 ml. over time and splitting the sample into two separate immobilized enzyme nylon tube reactors. The hydrogen peroxide produced by enzymatic reaction was detected by the hydrogen peroxide electrode. After the analog digital conversion, the concentrations of glucose and glutamate in mgrdl were shown by the printer. Whenever the glucose concentration of the fermentation broth is below the preset value, the computer can start the feedback control unit to add glucose to the fermentor and keep the glucose concentration at the preset value. In the system there is a calibration unit that can be started by the computer to recalibrate the system with standard glucose and glutamate solutions Ž100 mgrdl glucose and 50 mgrdl glutamate. after every 20 measurements.
Ž 4. The amounts of hydrogen peroxide produced by reactions Ž3. and Ž4. are respectively proportional to the amount of glucose in reaction Ž3. and glutamate in reaction Ž4.. Thus, by measuring the hydrogen peroxide produced, one can determine the content of glucose and glutamate in assayed solutions. 3.1.2. Preparation of on-line monitoring system Glucose and glutamate oxidases were covalently coupled to the inner side of a nylon tube by glutaraldehyde as described by Sundaram and Hornby Žwith some modifications. w8x, to construct the nylon tube reactors. Fig. 4 shows the diagram of the system. The on-line monitoring system consists of the following: sampling unit, electronic micromotor-driven pumps, dilution unit, injection valve,
3.1.3. Linear range of the working curÕe For determination of glucose, the response of the system was linear up to 600 mgrdl, assayed in 50 mmolrl, pH 5.5 phosphate buffer. For measurement of glutamate, the linear range of working curve was 10–60 mgrdl, assayed in 50 mmolrl, pH 7.0 phosphate buffer. 3.1.4. Reproducibility of the system The variation coefficients for 20 performance-responses to 100, 400 and 1000 mgrdl of glucose were found to be 2.96%, 2.40% and 3.2%, respectively. The response sensitivity of the system to glucose was 4.2–4.4 mVrmgrdl. The within-batch CV% values for 20 performance-responses to 10, 40 and 100 mgrdl of glutamate were found to be 6.27%, 2.34% and 2.62%, respectively. The response sensitivity was 5.6–8.8 mVrmgrdl.
Fig. 4. Diagram of the automatic on-line monitoring system.
J. Liu, G. Li r Sensors and Actuators B 65 (2000) 26–31
29
Fig. 5. Time course of the fermentation process.
3.1.5. Stability of the immobilized enzyme reactor One immobilized GOD nylon tube reactor, which worked in 50 mmolrl, pH 5.5 phosphate buffer and stored in the same buffer at 48C, could be used for more than 1100 assays. One immobilized glutamate oxidase reactor, which worked in 50 mmolrl, pH 7.0 phosphate buffer and stored in the same buffer at 48C, could be used for more than 2000 assays. 3.1.6. Fermentation process monitoring The feasibility study was performed using the on-line monitoring system to monitor and control the glutamate fermentation process of Corynebacterium crenatum AS 1.542 w9x in 200-l fermentor scale. The time course of the fermentation process ŽFig. 5. showed that the period of 0–15 h was the stationary stage. After this stage, the bacterial cell went into the logarithmic growth stage but the consumption of glucose and the production of glutamate were behind the cell growth. After about 40 h of fermentation, the glucose level was down from 11% to 5%, and the glutamate level was up from 0% to 4%. The enzymatic kit of glucose determination was used in parallel, for a comparison test. If X shows the values assayed
Fig. 7. The on-line monitoring system used for on-line feedback control of glucose in glutamate-fermentation process.
by the kit method and Y shows the values determined by the system, the regression equation is Y s 0.348 q 0.9617X and the correlation coefficient is r s 0.9794 ŽFig. 6.. 3.1.7. Feedback control of glucose leÕel Continuous monitoring and feedback controlling of glucose level have been performed in another batch fermentation process using the system. The fermentation process started at a glucose level of 1000 mgrdl. After about 17 h of fermentation, the glucose level was down to the preset value Ž300 mgrdl.. Then, the feedback control unit was activated to add the feedback solution Ž2000 mgrdl. to the fermentor to keep the glucose level around the preset value; the maximum error of the feedback control was less than 5% ŽFig. 7.. Since there is a dilution unit in the system, it can perform feedback control at different preset values with corresponding dilution scales. 4. A batch-type enzyme field effect transistor (ENFET) sensor Penicillin-G concentration is a very important parameter in the penicillin fermentation process. The previous report w10x described an Hq-ISFET-type sensor based on penicillinase for determination of penicillin. However, because of the specificity of the enzyme, it is impossible to determine the real concentration of penicillin G in the fermentation broth. Now, a penicillin-G-sensitive FET-type sensor based on penicillin-G acylase and an Hq ISFET is introduced. 4.1. Experiments and results 4.1.1. Principle of the measurement The measurement is based on the following reaction:
™
penicillin-G acylase
penicillin G q H 2 O Fig. 6. Correlation and regression line of two methods.
6-aminopenicillanic acid q phenylacetic acid
30
J. Liu, G. Li r Sensors and Actuators B 65 (2000) 26–31
The variation of Hq ion concentration during the reaction is detected by the Hq ISFET. 4.1.2. Preparation of the penicillin G-sensitiÕe sensor system The differential Hq-ISFET chip, the dual Hq-ISFET tubes and a common reference electrode were fabricated and constructed at the Institute of Semiconductors, Academia Sinica. The procedure of immobilization of penicillin-G acylase is as the previous report w11x. The differential Hq-ISFET chip with immobilized penicillin-G acylase membrane was connected to the PGP-type Bioion-sensitive FET differential voltage meter ŽInstitute of Semiconductor., thus the batch penicillin-G-sensitive HqISFET sensor system was constructed. 4.1.3. Response time The response of the sensor to different concentrations of penicillin G was fast. Usually within 30 s of initiation of the measurement, the output voltage reached the maximum value regardless of the concentration of substrate. 4.1.4. Linear range of the calibration curÕe In a 20 mmolrl phosphate buffer at pH 7.0, the sensor was tested to measure the solutions containing different contents of penicillin G. The linear range of the calibration curve was 0.5–8 mmolrl. 4.1.5. Reproducibility of the sensor Three samples with different concentrations of penicillin G were measured by the sensor in a pH 7.0, 20 mmolrl phosphate buffer. Each sample was assayed repeatedly for 20 times. The values of the coefficient of variation of the outputs were all below 5%.
Fig. 9. Comparison of the values assayed by the sensor with values assayed by HPLC. The fermentation broth was assayed in a 20 mmolrl pH 7.0 phosphate buffer system. X values assayed by HPLC, Y values assayed by the sensor. Y s1.034 X y2083.7 Ž r s 0.9944..
4.1.6. Stability of the sensor probe (differential H q-ISFET chip with immobilized penicillin-G acylase) Stored in a 20 mmolrl phosphate buffer at pH 7.0 at 48C and periodically measuring the 5 mmolrl penicilin-G solution at room temperature, three sensor probes were stable for 6 months without a significant decrease of output value ŽFig. 8.. During this time, the three sensor probes were continually used for more than 1000 runs. 4.1.7. Measurement of penicilin-G content in fermentation broth The sensor system was tested in the determination of the penicillin-G content in broth during the penicilin fermentation process at North China Pharmaceutical factory. A total of 40 samples with low and high concentrations of penicillin G were chosen for the correlation test. Every sample was determined in parallel with the high-pressure liquid chromatography method ŽHPLC; Shimadzu. using the same standard. Comparing the values determined by the HPLC method with that of the values assayed by the sensor method, these two methods had a good coincidence ŽFig. 9.. The correlation coefficient was r s 0.9944 and the regression equation was Y s 1.034 X y 2083.7 Ž X values assayed by the HPLC method; Y values assayed by the sensor method.. 5. Conclusions
Fig. 8. The stability of the sensor probes.
A biosensor based on chemiluminescence for serum uric acid determination and two sensors for fermentation process monitoring and controlling have been developed. All the sensors have good properties and long-term opera-
J. Liu, G. Li r Sensors and Actuators B 65 (2000) 26–31
tional stability. The three sensor methods have a good coincidence with routinely used methods.
Acknowledgements Financial support for the study was provided by the Chinese National ‘‘8.5’’ Science and Technology Development Program, and partially by the State Key Laboratory of Transducer Technology.
References w1x G.X. Li, Y. Shao, J.G. Liu, G.S. Wang, Use of immobilized uricase electrode in uric acid analysis, Biosensors ’90 Ž1990. 297. w2x M.T. Reilly, M. Charles, The use of on-line sensors in bioprocess control, in: Sensors in Bioprocess Control, Marcel Dekker, New York, 1990, pp. 243–291. w3x R.J. Geise, A.M. Yacynych, Electrochemical biosensors for bioprocess control, in: Sensors in Bioprocess Control, Marcel Dekker, New York, 1990, pp. 173–191. w4x J. Bradley, T. Ding, W. Vahjen, S. White, U. Bilitewski, E. D’Costa, W. Stamm, J. Higgins, R.D. Schmid, Biosensors for fermentation control, in: Biosensors: Fundamentals, Technologies, and Applications, Verlag Chemie, Weinheim, 1992, p. 209. w5x D.T. Bostick, D.M. Hercules, Quantitative determination of blood glucose using enzyme induced chemiluminescence of luminol, Anal. Chem. 47 Ž1975. 447–452.
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w6x J.G. Liu, J. Guo, G.X. Li, Biosensor based on chemiluminescence for serum uric acid determination, Chin. J. Biotechnol. 11 Ž3. Ž1995. 177–183. w7x M. Tabata, C. Fukunaga, M. Ohyabu, T. Murachi, Highly sensitive flow injection analysis of glucose and uric acid in serum using an immobilized enzyme column and chemiluminescence, J. Appl. Biochem. 6 Ž1984. 251–258. w8x P.V. Sundaram, W.E. Hornby, FEBS Lett. 10 Ž1970. 325–327. w9x Q. Chen, L. Li, Studies on L-glutamic acid producing bacteria AS 1.542, Acta Microbiol. Sin. 16 Ž1976. 37–40. w10x L.C. Zhong, G.X. Li, Biosensor based on ISFET for penicillin determination, Sens. Actuators, B 13–14 Ž1993. 570. w11x J.G. Liu, L. Liang, G.X. Li, R.S. Han, K.M. Chen, Hq-ISFET-based biosensor for determination of penicillin G, Biosens. Bioelectron. Ž1998. Žin press..
Biographies Jianguo Liu is an Associate Professor at the Institute of Microbiology of the Chinese Academy of Sciences. Liu obtained a Bachelor of Science degree from Nanjing Agricultural University in 1982. Liu’s current fields of interest include enzymology, molecular biology, enzyme engineering, and biosensors.
Gaoxiang Li is a Professor at the Institute of Microbiology of the Chinese Academy of Sciences. Li earned a degree in Bachelor of Science from Beijing University in 1957. Li’s current fields of interest include enzymology, enzyme engineering, and biosensors.
Application of biosensors for diagnostic analysis and bioprocess monitoring Jianguo Liu ) , Gaoxiang Li Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People’s Republic of China State Key Laboratory of Transducer Technology, People’s Republic of China Received 30 July 1998; accepted 8 January 1999
Abstract A biosensor based on chemiluminescence for determination of serum uric acid and two biosensors for glutamate- and penicillin-fermentation process monitoring and controlling have been developed. The preparations and some properties of these sensors have been described in detail. The sensor methods described have been compared with routine methods used and the good coincidences have been obtained. q 2000 Elsevier Science S.A. All rights reserved. Keywords: Uric acid sensor; Chemiluminescence; Glutamate fermentation process control; ENFET sensor
1. Introduction The serum uric acid concentration is an important index for clinical diagnosis of gout, leukemia toxemia of pregnancy and severe renal impairment. In the enzyme electrode research w1x, we found out that the sensitivity of the O 2 electrode did not meet the demand of serum uric acid measurement, especially when the concentration was below 3 mgrdl. The high sensitivity of luminol chemiluminescence can overcome this drawback. Biosensors for fermentation process control hold much promise for monitoring many of the important compounds involved in fermentation. These include glucose, glutamate, ethanol, organic acid, and various gases w2–4x. Recently, two biosensors for fermentation process control have been developed in our laboratory. One is the model on-line monitoring system and the other is the batch-type ENFET sensor. This paper introduces three biosensors developed in our laboratory recently.
)
Corresponding author. Fax: q86-10-62560912.
2. A chemiluminescence sensor for serum uric acid assay 2.1. Experiments and results 2.1.1. Principle of the assay The method was based on following reaction sequence: uric acid q O 2
™
uricase
allantoinq H 2 O 2 q CO 2
™
Ž 1.
ferricyanide
H 2 O 2 q luminol
aminophthalate anion q photonq N2 q 2H 2 O
Ž 2.
Reaction Ž1. is highly specific for uric acid. However, reaction Ž2. is catalyzed by enzyme Žperoxidase. or metal ions possessing an oxidation state w5x. Provided all the conditions are constant, the emitted photon intensity is proportional to uric-acid concentration in the serum. 2.1.2. Preparation of the chemiluminescent sensor Uricase ŽEC 1.7.3.3. was immobilized on the alkylamined pore glass with glutaraldehyde w6x. The immobilized enzyme was packed in a small plastic tube to build the enzyme reaction column. Fig. 1 indicates the flowthrough diagram of the sensor. A certain volume of serum sample met the carrier buffer through the valve injector,
0925-4005r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved. PII: S 0 9 2 5 - 4 0 0 5 Ž 9 9 . 0 0 4 3 1 - 1
J. Liu, G. Li r Sensors and Actuators B 65 (2000) 26–31
27
Fig. 1. Diagram of FIA for determination of uric acid using uricase-chemiluminescence sensor system.
then flowed into the immobilized enzyme reactor column. After enzymatic reaction, the mixture joined the luminol and ferricyanide solution in the reaction cell. The photon generated by chemiluminescent reaction was detected by a photodiode attached to the face of the reaction cell. The photon intensity was shown on the window of the photoelectric generator. 2.1.3. Response time The response time of the sensor to various concentrations of serum uric acid with a 17-ml sample volume was short. The photon intensity of the reaction reached its peak value about 47 s after injection. It required about 1.5 min for every sample measurement. 2.1.4. Linearity of the working curÕe Typical linearity of the working curve was from 1 up to 20 mgrdl Ž r s 0.9999; Fig. 2.. This linear range was wider than that obtained by the electrochemical electrode method w1x and the result reported by Tabata et al. w7x. 2.1.5. Precision and reproducibility Pooled serum samples with low and high contents of uric acid were analyzed 20 times Žwithin a day. or 20 times for 10 days Žday to day.. The imprecision within a
Fig. 2. Calibration curve for uric acid determination using uricase-chemiluminescence sensor system.
day was below 5%, and the day-to-day imprecision was below 8%, which were satisfactorily precise and reproducible for clinical analysis. 2.1.6. Operational stability The operational stability of the sensor depends on the stability of the immobilized uricase column. Used at room temperature Ž25–328C. and stored at 48C, the immobilized uricase column retained 94% of its original activity even after 2000 runs for 5.5 months of continual usage. No decrement of the photon intensity was observed. 2.1.7. Comparison with other method Sixty-one serum samples of high and low uric acid content were measured by the present method, and the standard colorimetric method routinely used in clinical labs employing the enzymatic kit ŽShanghai, China. performed on a Hitachi 7150 autoanalyzer. As shown in Fig. 3, excellent agreement was obtained. The calculated regression line and correlation coefficient were Y s 0.4 q 0.938 X Ž X data measured by enzymatic kit; Y data measured by present method. and r s 0.9909, respectively.
Fig. 3. Correlation and regression line for serum uric acid measurement assayed by uricase-chemiluminescence-sensor method against enzymatic kit method. X, data assayed by enzymatic kit method. Y, data assayed by the sensor method.
J. Liu, G. Li r Sensors and Actuators B 65 (2000) 26–31
28
3. An on-line monitoring system The purpose of this study was to construct a model on-line system for fermentation process control. The on-line monitoring of glutamate fermentation process was chosen as the target of the study. During the fermentation process, the concentration of glucose Žsubstrate. and glutamate Žproduct. are very important parameters, which reflect the development of the fermentation process. 3.1. Experiments and results 3.1.1. Principle of the measurement The measurement is based on the following reaction sequences:
™
glucose oxidase
Glucoseq O 2 q H 2 O
Glucono-D-lactone q H 2 O2
™
Ž 3.
glutamate oxidase
2-L-glutamateq O 2 q H 2 O
2-oxoglutarate q 2NH 3 q H 2 O 2
nylon tube reactors, hydrogen peroxide electrode, analog– digital converter, recorder and feedback control unit. Driven by the electronic micromotor pump, the fermentation broth passes through the sampling unit, which prevents the microorganisms and insoluble materials from passing into the downstrain analytic system, and flows through the dilution unit and the injection valve, ensuring the same sample volume Ž15 ml. over time and splitting the sample into two separate immobilized enzyme nylon tube reactors. The hydrogen peroxide produced by enzymatic reaction was detected by the hydrogen peroxide electrode. After the analog digital conversion, the concentrations of glucose and glutamate in mgrdl were shown by the printer. Whenever the glucose concentration of the fermentation broth is below the preset value, the computer can start the feedback control unit to add glucose to the fermentor and keep the glucose concentration at the preset value. In the system there is a calibration unit that can be started by the computer to recalibrate the system with standard glucose and glutamate solutions Ž100 mgrdl glucose and 50 mgrdl glutamate. after every 20 measurements.
Ž 4. The amounts of hydrogen peroxide produced by reactions Ž3. and Ž4. are respectively proportional to the amount of glucose in reaction Ž3. and glutamate in reaction Ž4.. Thus, by measuring the hydrogen peroxide produced, one can determine the content of glucose and glutamate in assayed solutions. 3.1.2. Preparation of on-line monitoring system Glucose and glutamate oxidases were covalently coupled to the inner side of a nylon tube by glutaraldehyde as described by Sundaram and Hornby Žwith some modifications. w8x, to construct the nylon tube reactors. Fig. 4 shows the diagram of the system. The on-line monitoring system consists of the following: sampling unit, electronic micromotor-driven pumps, dilution unit, injection valve,
3.1.3. Linear range of the working curÕe For determination of glucose, the response of the system was linear up to 600 mgrdl, assayed in 50 mmolrl, pH 5.5 phosphate buffer. For measurement of glutamate, the linear range of working curve was 10–60 mgrdl, assayed in 50 mmolrl, pH 7.0 phosphate buffer. 3.1.4. Reproducibility of the system The variation coefficients for 20 performance-responses to 100, 400 and 1000 mgrdl of glucose were found to be 2.96%, 2.40% and 3.2%, respectively. The response sensitivity of the system to glucose was 4.2–4.4 mVrmgrdl. The within-batch CV% values for 20 performance-responses to 10, 40 and 100 mgrdl of glutamate were found to be 6.27%, 2.34% and 2.62%, respectively. The response sensitivity was 5.6–8.8 mVrmgrdl.
Fig. 4. Diagram of the automatic on-line monitoring system.
J. Liu, G. Li r Sensors and Actuators B 65 (2000) 26–31
29
Fig. 5. Time course of the fermentation process.
3.1.5. Stability of the immobilized enzyme reactor One immobilized GOD nylon tube reactor, which worked in 50 mmolrl, pH 5.5 phosphate buffer and stored in the same buffer at 48C, could be used for more than 1100 assays. One immobilized glutamate oxidase reactor, which worked in 50 mmolrl, pH 7.0 phosphate buffer and stored in the same buffer at 48C, could be used for more than 2000 assays. 3.1.6. Fermentation process monitoring The feasibility study was performed using the on-line monitoring system to monitor and control the glutamate fermentation process of Corynebacterium crenatum AS 1.542 w9x in 200-l fermentor scale. The time course of the fermentation process ŽFig. 5. showed that the period of 0–15 h was the stationary stage. After this stage, the bacterial cell went into the logarithmic growth stage but the consumption of glucose and the production of glutamate were behind the cell growth. After about 40 h of fermentation, the glucose level was down from 11% to 5%, and the glutamate level was up from 0% to 4%. The enzymatic kit of glucose determination was used in parallel, for a comparison test. If X shows the values assayed
Fig. 7. The on-line monitoring system used for on-line feedback control of glucose in glutamate-fermentation process.
by the kit method and Y shows the values determined by the system, the regression equation is Y s 0.348 q 0.9617X and the correlation coefficient is r s 0.9794 ŽFig. 6.. 3.1.7. Feedback control of glucose leÕel Continuous monitoring and feedback controlling of glucose level have been performed in another batch fermentation process using the system. The fermentation process started at a glucose level of 1000 mgrdl. After about 17 h of fermentation, the glucose level was down to the preset value Ž300 mgrdl.. Then, the feedback control unit was activated to add the feedback solution Ž2000 mgrdl. to the fermentor to keep the glucose level around the preset value; the maximum error of the feedback control was less than 5% ŽFig. 7.. Since there is a dilution unit in the system, it can perform feedback control at different preset values with corresponding dilution scales. 4. A batch-type enzyme field effect transistor (ENFET) sensor Penicillin-G concentration is a very important parameter in the penicillin fermentation process. The previous report w10x described an Hq-ISFET-type sensor based on penicillinase for determination of penicillin. However, because of the specificity of the enzyme, it is impossible to determine the real concentration of penicillin G in the fermentation broth. Now, a penicillin-G-sensitive FET-type sensor based on penicillin-G acylase and an Hq ISFET is introduced. 4.1. Experiments and results 4.1.1. Principle of the measurement The measurement is based on the following reaction:
™
penicillin-G acylase
penicillin G q H 2 O Fig. 6. Correlation and regression line of two methods.
6-aminopenicillanic acid q phenylacetic acid
30
J. Liu, G. Li r Sensors and Actuators B 65 (2000) 26–31
The variation of Hq ion concentration during the reaction is detected by the Hq ISFET. 4.1.2. Preparation of the penicillin G-sensitiÕe sensor system The differential Hq-ISFET chip, the dual Hq-ISFET tubes and a common reference electrode were fabricated and constructed at the Institute of Semiconductors, Academia Sinica. The procedure of immobilization of penicillin-G acylase is as the previous report w11x. The differential Hq-ISFET chip with immobilized penicillin-G acylase membrane was connected to the PGP-type Bioion-sensitive FET differential voltage meter ŽInstitute of Semiconductor., thus the batch penicillin-G-sensitive HqISFET sensor system was constructed. 4.1.3. Response time The response of the sensor to different concentrations of penicillin G was fast. Usually within 30 s of initiation of the measurement, the output voltage reached the maximum value regardless of the concentration of substrate. 4.1.4. Linear range of the calibration curÕe In a 20 mmolrl phosphate buffer at pH 7.0, the sensor was tested to measure the solutions containing different contents of penicillin G. The linear range of the calibration curve was 0.5–8 mmolrl. 4.1.5. Reproducibility of the sensor Three samples with different concentrations of penicillin G were measured by the sensor in a pH 7.0, 20 mmolrl phosphate buffer. Each sample was assayed repeatedly for 20 times. The values of the coefficient of variation of the outputs were all below 5%.
Fig. 9. Comparison of the values assayed by the sensor with values assayed by HPLC. The fermentation broth was assayed in a 20 mmolrl pH 7.0 phosphate buffer system. X values assayed by HPLC, Y values assayed by the sensor. Y s1.034 X y2083.7 Ž r s 0.9944..
4.1.6. Stability of the sensor probe (differential H q-ISFET chip with immobilized penicillin-G acylase) Stored in a 20 mmolrl phosphate buffer at pH 7.0 at 48C and periodically measuring the 5 mmolrl penicilin-G solution at room temperature, three sensor probes were stable for 6 months without a significant decrease of output value ŽFig. 8.. During this time, the three sensor probes were continually used for more than 1000 runs. 4.1.7. Measurement of penicilin-G content in fermentation broth The sensor system was tested in the determination of the penicillin-G content in broth during the penicilin fermentation process at North China Pharmaceutical factory. A total of 40 samples with low and high concentrations of penicillin G were chosen for the correlation test. Every sample was determined in parallel with the high-pressure liquid chromatography method ŽHPLC; Shimadzu. using the same standard. Comparing the values determined by the HPLC method with that of the values assayed by the sensor method, these two methods had a good coincidence ŽFig. 9.. The correlation coefficient was r s 0.9944 and the regression equation was Y s 1.034 X y 2083.7 Ž X values assayed by the HPLC method; Y values assayed by the sensor method.. 5. Conclusions
Fig. 8. The stability of the sensor probes.
A biosensor based on chemiluminescence for serum uric acid determination and two sensors for fermentation process monitoring and controlling have been developed. All the sensors have good properties and long-term opera-
J. Liu, G. Li r Sensors and Actuators B 65 (2000) 26–31
tional stability. The three sensor methods have a good coincidence with routinely used methods.
Acknowledgements Financial support for the study was provided by the Chinese National ‘‘8.5’’ Science and Technology Development Program, and partially by the State Key Laboratory of Transducer Technology.
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Biographies Jianguo Liu is an Associate Professor at the Institute of Microbiology of the Chinese Academy of Sciences. Liu obtained a Bachelor of Science degree from Nanjing Agricultural University in 1982. Liu’s current fields of interest include enzymology, molecular biology, enzyme engineering, and biosensors.
Gaoxiang Li is a Professor at the Institute of Microbiology of the Chinese Academy of Sciences. Li earned a degree in Bachelor of Science from Beijing University in 1957. Li’s current fields of interest include enzymology, enzyme engineering, and biosensors.